Both viruses and non-viral gene transfer vectors must overcome a series of cellular barriers to transduce or transfect successfully a cell. An important hurdle is endosomal escape. The study of endosomal escape of viruses on a molecular level is complicated by the inherent complexity of viruses that results from structural constraints and constraints imposed by additional viral functions such as viral exit. In contrast, the analysis of peptide mediated endosomal escape offers a minimal system that allows the analysis of this barrier on a molecular level. In a first step, we will systematically explore the sequence requirements for peptides that have the ability to enhance endosomal escape. Careful analysis of these sequences will likely help to identify endosome escape sequences in viruses. We will use a well-defined "minimal virus" - a non-viral vector - to test the efficacy of these peptides in tissue culture. A diverse set of such peptides will also allow us to explore the scope of different molecular mechanisms of endosomal escape. Because viruses likely use identical or very similar mechanisms, this will improve our understanding of the mechanisms of viral endosome escape as well as lead to improved non-viral vectors. We already identified a collection of such peptides, at least one of which shows lysogenic and fusogenic properties in a pH-dependent fashion. To test its potency in tissue culture we incorporated this peptide into a non-viral vector and were able to demonstrate that it leads to a roughly 100-fold enhancement of transfection efficiencies. We will measure quantitatively the escape of non-viral vector DNA from the endosomal pathway into the cytoplasm. Mechanistic studies using the newly identified peptides are aimed at determining the point of endosomal escape (early vs late endosomes) as well as the molecular mechanism of endosome lysis or fusion of endosomal and liposomal membranes. These studies will include the determination of the exclusion limits of the peptide-induced pores both by in vitro assays using liposomes as well as by studies by fluorescence microscopy in intact cells. The role of the nature of the lipid anchor in peptide mediated fusion will be studied using lipid anchors that have differential effects in SNARE mediated fusion.